中国麻风皮肤病杂志 ›› 2018, Vol. 34 ›› Issue (1): 16-20.

• 论著 • 上一篇    下一篇

以分泌型荧光素酶为报告基因的Nrf2报告系统的建立

张尔婷1,2 江 娜1,2 刘 清1,2 彭丽倩1,2 张倩雯1 梁碧华1 李润祥1 邓蕙研1 李华平1 朱慧兰1   

  1. 1.广州市皮肤病防治所，广东 广州 510095 2.广州医科大学，广东 广州510182
  • 出版日期:2018-01-15 发布日期:2018-12-05
  • 通讯作者: 朱慧兰，E-mail：zhlhuilan2126.com
  • 基金资助:
    广州市2014年自然科学基金

Establishment of a Nrf2 reporting system based on secreted luciferase reporter gene

ZHANG Erting1,2, JIANG Na1,2, LIU Qing1,2, PENG Liqian1,2, ZHANG Qianwen1, LIANG Bihua1, LIRunxiang1, DENG Huiyan1, LI Huaping1, ZHU Huilan1.   

  1. 1.Guangzhou Institute of Dermatology, Guangzhou 510095, China 2. Guangzhou Medical University, Guangzhou 511436, China
  • Online:2018-01-15 Published:2018-12-05
  • Contact: ZHU Huilan, E-mail:zhlhuilan@126.com

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摘要： 目的：建立以分泌型荧光素酶Gaussia Luciferase (Gluc)为报告基因的Nrf2信号通路的报告系统。方法：以Gluc为报告基因，构建以ML-ARE和CV-ARE为基础的转录序列。以ML，CV启动子驱动的碱性磷酸酶（Alkinane Phosphtase, AP）为内参报告，在EcoRI---XhoI之中分别串联1个，2个，4个，8个ARE元件，比较含ARE元件不同数目的转录序列元件下Gluc的活性。结果：含有8个ARE元件的ML-ARE和CV-ARE转录序列的Gluc活性最大；ML-ARE较CV-ARE对抗氧化活性的干扰较小；ML-8XARE质粒转染至HaCaT细胞后，予莱菔硫烷(SF)和特丁基对苯二酚(BHQ)处理6h、UV辐射后4h时，Gluc活性最大。 结论：本研究成功构建了以Gluc为报告基因的Nrf2报告系统，该系统为UV疾病细胞模型的建立奠定了基础。

关键词: 报告系统, Nrf2, Gluc

Abstract: Objective: To establish the Nrf2 signaling pathway activation reporting system with secreted Gaussia luciferase (Gluc) as report gene. Methods: The transcribed sequences based on CV-ARE and ML-ARE, Gluc as a reporter gene, were constructed. Alkaline phosphatase (AP) promoted by ?ML and CV promoter as an internal report, different numbers (1, 2, 4 or 8) of ARE were inserted into EcoRI---XhoI respectively. The activity of Gluc with different numbers of ARE were compared. Results: The fluorescence activity of Gluc with ML-8XARE and CV-8XARE was the highest.. The interference of ML-ARE to antioxidant was lower than CV-ARE. The ML-8XARE plasmid was transfect to HaCaT cells, then were treated by sulforaphane (SF) and tert butyl hydroquinone (TBHQ) or UV. The activity of Gluc was the highest at 6 hours after treated by SF and TBHQ and at 24 hours radiated by UV. Conclusion: The Nrf2 signaling pathway activation reporting system with secreted Gaussia luciferase (Gluc) as report gene was successfully constructed, which lay the foundation for the model of UV disease.

Key words: reporting system, Nrf2, Gluc